We propose to investigate the metabolism of the phthalic acids and their esters by soil and water bacteria. The immediate objectives are: (a) to establish the catabolic routes used by bacteria to degrade these phthalates, (b) to study the hydroxylase(s) involved in the transformation of o-phthalate to 4,5-dihydroxyphthalate and characterize the initial intermediate(s), i.e., to determine whether a dihydrodiol or a monohydroxy derivative is formed first, (c) to purify and characterize 4,5-dihydroxyphthalate decarboxylase, the non-oxidative decarboxylation step in the o-phtlalate pathway of Ps. putida, and (d) to provide information on the biodegradability of the phthalate esters that are commonly used and have appeared as pollutants of the environment. The information obtained from these four specific objectives will be related to the well established catabolic sequences used by bacteria for the degradation of aromatic compounds. The metabolic pathways will be determined by the accumulation and identification of intermediates by whole cells and extracts; the respiratory patterns of induced and non-induced cells; isolation of mutants blocked in the pathways to prepare early intermediates. Phthalate hydroxylase and 4,5-dihydroxyphthalate decarboxylase will be purified by standard chromatographic procedures and the proteins examined for subunit structure, cofactor requirements, catalytic activities and specificity. The biodegradability of the phthalate esters will be examined with enrichment cultures and perfusion systems, and the esterase activities of these, and pure typed cultures will be analyzed for phthalate ester hydrolysis. BIBLIOGRAPHIC REFERENCES: Keyser, P. and Ribbons, D. W. (1975). Bact. Proc. I60. "o-Phthalate Oxygenase Activity from Pseudomonas fluorescens." Pujar, B. G. and Ribbons, D. W. (1976). Bact. Proc. (in press). "Purification of 4,5-Dihydroxyphthalate Decarboxylase and Protocatechuate 3,4-Oxygenase by Affinity Chromatography."